Vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). The dramatic and ongoing pandemic attributed to the MBFV Zika virus is important because of its capacity to cause a severe congenital Zika disease as well an ever increasing spectrum of neurological syndromes in adults. Our current research is focused on the TBFV, but studying the biology of TBFV will elucidate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We primarily study LGTV, a naturally attenuated member of the TBFV that can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. In addition, we also study the virulent autochthonous BSL-3 POWV/DTV. Neither of these 2 agents are Select Agents which greatly facilitate research studies. In addition, we have also begun to study the BSL2 MBFV, Zika virus. With the recent emergence of Zika in Latin America and the US, similar avenues of inquiry are being explored for that vexatious pathogen. Comparison of VBFV cytoarchitecture in mammalian and arthropod cells. We previously published a study comparing LGTV virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. This last year, we published similar studies with ZIKV in a human neuroblastoma cell line (SK-N-SH) and mosquito cell line C6/36 from A. albopictus. ZIKV replicated well in SK-N-SH and C6/36. ZIKV caused an acute lytic crisis accompanied by frank apoptosis in SK-N-SH cells, but the effects of infection in C6/36 cells were imperceptible despite the fact that activated caspase 3 staining was readily apparent. These findings mirrored those we have seen with the TBFV. The results of ultrastructural studies on SK-N-SH and C6/36 cells revealed that the cytoarchitecture of ZIKV infection was similar to that seen previously with the TBFV. However, the application of dual-tilt electron tomography lead to resolution substantially better than our previous work. Zika virus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. Differences between infected SK-N-SH and C6/36 were less prominent than those seen with TBFV. A Molecular biology and molecular pathogenesis of acute and persistent VBFV infection. The role of persistent infection in natural life cycle of TBFV in rodent and arthropod hosts is not well characterized, but may also be responsible for prolonged debilitating sequelae observed in survivors of acute TBFV infection. Experimental work by others on MBFV and the recent findings that ZIKV can persist in human semen and other sites for extended periods of time strongly imply that persistence of MBFV may also be an underappreciated feature of those infections. In the past year, we published our studies of TBFV persistence to inspect the cellular transcriptome during the initiation and maintenance of persistent infection. As noted, LGTV infection causes an acute lytic crisis of most cells, and we have shown the mechanism of cell death is apoptosis. The surviving cells somehow evade apoptosis and go on to establish persistent infection. Our agnostic deep sequencing approach to the study of cellular transcriptomics confirmed that gene pathways associated with cell survival and apoptosis avoidance were involved. A signature of 451 genes was associated with the initiation of persistence. Intensive bioinformatics perusal using Ingenuity Pathway Analysis and other similar software packages, revealed that networks associated with cell survival were playing a role. The acute phase was associated with networks and pathways. We also unpublished our novel deep sequencing approach to examine the viral genomics during TBFV infection in tick cells (ISE-6). ISE-6 cells develop a persistent infection with no apparent acute phase and there is no evidence of DI particles being involved in the initiation or maintenance of persistence. A transcriptomic analysis of infected ISE6 cells is being prepared for publication. TBFV infection in the reservoir host. Peromyscus leucopus is the identified reservoir host for POWV. Parallel POWV infection of Permomyscus and Balb/C and C57Bl/6 mice yielded surprising results which we published this year. POWV causes a fulminant and lethal neurological infection in both strains of lab mice after either intracerebral or intraperitoneal infection. However, the Peromyscus mice show no overt signs of disease after either ic or ip inoculation. Detailed studies reveal that no evidence of POWV infection can be detected after ip inoculation of Peromyscus mice and, furthermore that after ic infection, virus replication is severely restricted in time and place to the olfactory bulb and projecting tracts. Only a short-lived and very low level viremia occurs but a neutralizing antibody response does develop. The mechanism for the virus restriction is being pursued in vitro and in commercially available mouse embryo cell lines from B6 and Peromyscus.